PROJECT SUMMARY The project goal is to understand the selective pressures associated with Zika virus (ZIKV) adaptation. New approaches to preventing ZIKV are needed because the endemic range of this virus is expanding and because current methods are limited to controlling mosquito populations, and they cannot prevent invasion of this virus into new locales. A promising candidate for arbovirus control and prevention relies on the introduction of the intracellular bacterium Wolbachia into Ae. aegypti mosquitoes. Wolbachia biocontrol has advanced from laboratory experiments demonstrating that Wolbachia reduces the transmission potential of Ae. aegypti for certain viruses to small-scale field trials demonstrating that Wolbachia are capable of spreading through wild Ae. aegypti populations. This primarily has been proposed as a tool to control dengue virus (DENV) transmission; however, Wolbachia infections confer protection for their insect hosts against a range of pathogens including for Ae. aegypti against ZIKV. A critical next step is to assess the impact of Wolbachia on virus evolution to determine whether or not Wolbachia biocontrol is a sustainable method for ZIKV control and prevention that will not be undermined by mosquito or virus evolution. Likewise, alternative transmission pathways for ZIKV (e.g., sexual intercourse and perinatal transmission) are creating a scenario by which a highly mutable RNA virus could readily exploit new routes of transmission during circulation in South America. Accordingly, through this NIH/NIAD R21 we will perform experimental evolution studies to evaluate and anticipate evolutionary changes of ZIKV- Wolbachia-mosquito interactions and vertebrate co-adaptation. Furthermore, Wolbachia-infected Ae. aegypti colonies have been established by a complementary Eliminate Dengue Program (EDP) project in Colombia to evaluate the impact of Wolbachia biocontrol on DENV transmission. There are two specific aims 1.) Evaluate the impact of Wolbachia-infected Ae. aegypti on ZIKV transmission potential and 2.) Evaluate the consequences of releasing ZIKV from alternate cycling between vertebrate and invertebrate hosts. Experimental evolution of ZIKV via alternate passage between Wolbachia-infected mosquitoes and mice is new. Medium-scale Wolbachia deployments have commenced or are imminent. Therefore, assessing the evolutionary potential of ZIKV to adapt to Wolbachia-infected Ae. aegypti will help inform the viability of Wolbachia biocontrol for ZIKV control. Potentially, through this R21, valuable evidence could be provided that justifies expanding this type of control program to other Ae. aegypti-transmitted arboviruses, e.g., ZIKV. And understanding the potential for new transmission pathways will be critically important for prediction, prevention, and control of this emerging viral disease.